Antiprotozoan activity of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus)

Nonspecific cytotoxic cells (NCC) obtained from channel catfish (Ictalurus punctatus) kill Tetrahymena pyriformis, an opportunistic parasite in fish. Based upon this fact, a new mechanism for nonspecific cellular anti-parasitic immunity in fish is proposed. Optimum in vitro conditions for NCC killing of deciliated T. pyriformis were first obtained. Lysis of T. pyriformis by NCC occurred by 10 hr of cocultivation of effector and target cells. During this time period, 50 to 60% cytotoxicity occurred. Fish anti-T. pyriformis serum enhanced NCC killing of T. pyriformis either by prolonging immobilization (after the cilia regeneration period) or by delaying cilia regeneration. Shared antigenic determinants between T. pyriformis, Ichthyophthirius multifiliis, and NC-37 target cells were demonstrated by binding-depletion experiments. For these studies, NCC were depleted from anterior kidney cells (the hemopoetic organ in fish) by preincubating formalin-treated T. pyriformis, I. multifiliis, or viable NC-37 target cells with NCC for 3 hr. Conjugates of effector and target cells were removed by overlaying on fetal bovine serum. Unconjugated fish anterior kidney cells were tested for cytotoxic activity against NC-37 or T. pyriformis target cells. Cold target inhibition experiments by using a 4-hr 51chromium cytotoxicity assay also demonstrated these shared antigenic determinants. Target-specific antisera, used to mediate the killing of T. pyriformis by NCC, were required only for immobilizing the targets, and did not function in an antibody-dependent cell-mediated (ADCC)-like mechanism. Scanning electron micrographs of NCC-T. pyriformis conjugates additionally demonstrated NCC binding to both cilia and cell surface determinants.     

The ultrastructural characteristics of the interaction of Legionella pneumophila with the infusorian protozoon Tetrahymena pyriformis

In this work the morphological features of the interaction of L. pneumophila virulent strain and T. pyriformis have been studied on the submicroscopic level in the time course of the process. The study has shown the process of the destruction of the bacterial population and the penetration of individual intact Legionella cells from the phagosome into the endoplasm of T. pyriformis after 6-9 hours of interaction in the form of the budding of the phagosome and further multiplication of Legionella in the endoplasm. As revealed in this study, T. pyriformis have two types of phagosomes characterized by different variants of the destruction of Legionella. In T. pyriformis lysosomes-like granules, mitochondria and the granular endoplasmatic network take part in the process of interaction. The process of interaction has been found to end by day 7 in the death of all protozoal cells taking part in interaction.     

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.     

The ultrastructural characteristics of Francisella tularensis interaction with Tetrahymena pyriformis

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.     

Recognition of Prey by Suctoria: The Role of Cilia

ABSTRACT. The interaction between the predator, Discophrya collini , and the prey, Tetrahymena pyriformis , was investigated in order to determine what prey factors(s) may be recognized, facilitating the feeding response of the suctorian predator. When viable, deciliated T. pyriformis were offered to starved D. collini , only 12% of the suctoria captured a ciliate compared to 98% when presented with ciliated T. pyriformis . Starved suctoria were also offered three types of tanned sheep erythrocytes used as artificial prey. After exposure to erythrocytes coated with sonicates of T. pyriformis , 16% of the D. collini displayed one or more attached erythrocytes but after incubation with erythrocytes coated with T. pyriformis cilia, 72% of the suctoria had tentacles with attached cells. No untreated, tanned erythrocytes were captured. When T. pyriformis were treated with ciliary antiserum and then offered to starved D. collini , the resulting capture rate was low (13%), but T. pyriformis treated with normal rabbit serum were captured at a rate comparable to controls (92%). These results suggest that the prey's cilia and/or their surface macromolecules may facilitate prey capture.     

Species-specific antibodies of Tetrahymena acid alpha-glucosidase.

1. Tetrahymena acid alpha-glucosidases A and B were purified from T. pyriformis W and T. thermophila 399, respectively. The acid alpha-glucosidases A and B were different in immunological properties and thermostability. 2. The acid alpha-glucosidases A and B reflected specific distribution between T. pyriformis and T. thermophila. 3. Type A and B of taurolipid showed a species-specific distribution pattern between T. pyriformis and T. thermophila.     

Comparative Study of the Effect of Snake Venoms on the Growth of Ciliates Tetrahymena pyriformis: Identification of Venoms with High Antiprotozoal Activity

Doklady Biochemistry and Biophysics - To search for compounds with antiprotozoal activity, effects of snake venoms on the ciliates Tetrahymena pyriformis was studied. T. pyriformis from subkingdom...     

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species.

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.     

Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis.

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.     

Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.     

A cytophotometric study of the influence exerted by epidermal growth factor on RNA and protein synthesis in the ciliate Tetrahymena pyriformis

The influence of EGF (10(-8) M) on RNA and protein synthesis in the ciliate Tetrahymena pyriformis was studied. It was found that EGF stimulated RNA and proteins synthesis in T. pyriformis within 3 h after addition of EGF.     

Macronuclear chromatin of Blepharisma japonicum compared to that of Tetrahymena pyriformis

Abstract The macronuclear chromatin of the ciliate Blepharisma japonicum was studied by electrophoretic and immunological techniques as well as by micrococcal nuclease digestion and circular dichroism spectroscopy. Under these experimental conditions the macronuclear chromatin of B. japonicum was compared to that of Tetrahymena pyriformis . Data obtained through this analysis showed that the macronuclear chromatin of B. japonicum is structurally more relaxed than that of T. pyriformis . A perchloric acid soluble polypeptide, referred to as P3, is the only polypeptide of B. japonicum chromatin to appear immunologically related to the H1 histone of T. pyriformis . It is suggested that this P3 polypeptide in B. japonicum should be considered functionally equivalent to the T. pyriformis H1 histone, even though it differed from it both in terms of molecular mass and relative concentration.     

Infection of Tetrahymena pyriformis by Legionella longbeachae and other Legionella species found in potting mixes.

All Legionella longbeachae strains, both serogroups of L. bozemanii, and three strains of L. anisa reproducibly infected washed Tetrahymena pyriformis at 30 degrees C. L. pneumophila serogroup 1 strains infected T. pyriformis less reproducibly than did L. longbeachae. Low-level concentrations of nutrients in cocultures inhibited infection. Four L. micdadei strains and L. anisa ATCC 35292 failed to infect T. pyriformis.     

Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparations

Channel catfish, Ictalurus punctatus , were immunized with cilia from three isolates of Tetrahymena pyriformis and challenged with Ichthyophthirius multifiliis using a reproducible quantitative procedure. Two different methods of deciliation were used in antigen preparation. Results indicate that T. pyriformis cilia elicit an immune response in channel catfish against I. multifiliis , and that the protective ability of the cilia varies between T. pyriformis strains.     

Dissimilar effects of L and D amino acids on the growth of Tetrahymena

Of the L and D configurations of four amino acids (phenylalanine, valine, tryptophan, tyrosine) tested for influence on the growth rate of Tetrahymena, only L-tyrosine was able to induce imprinting in Tetrahymena pyriformis Zeuthen. D-valine stimulated the division of T.pyriformis NT-1, but failed to induce imprinting. The experiments have substantiated the selectivity of the amino acid receptors of Y.pyriformis, and the extraordinary imprinting potential of tyrosine as well, as judged by its influence on the growth rate.     

The effects of supraoptimal temperatures on population growth and cortical patterning in Tetrahymena pyriformis and Tetrahymena thermophila: a comparison

In this investigation, we compare the multiplication rates and morphogenetic responses of the two most studied Tetrahymena species, T. pyriformis and T. thermophila, at supraoptimal temperatures. Although the upper temperature limits differ greatly in the two species, the pattern of growth responses to high temperature is for the most part similar, with some differences in detail. The transient recovery of cell division at the highest temperature that allows cell division, characteristic of T. pyriformis, is observed in a less distinct form in T. thermophila. Moreover, there is a remarkable difference in developmental response, with drastic abnormalities in patterning of oral structures during the transient recovery of cell division in T. pyriformis, and far more limited abnormalities under similar conditions in T. thermophila. The abnormalities result from spatial disorder in the alignment and orientation of basal body pairs within the early oral primordium, followed by failures in the realignment that normally occurs as oral structures (membranelles and undulating membrane) mature. Both the initial spatial disorder and the failures in realignment are far more severe in T. pyriformis than in T. thermophila.     

Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

The arsenic metabolism in different biological organisms has been studied extensively. However, little is known about protozoa. Herein, we investigated the cell stress responses of the freshwater ciliate Tetrahymena pyriformis to arsenate toxicity. An acute toxicity assay revealed an 18-h EC(50) arsenate concentration of ca. 40 μM, which caused significant changes in the cell shape, growth and organism mobility. Whereas, under exposure to 30 μM arsenate, T. pyriformis could grow reasonably well, indicating a certain resistance of this organism. Arsenic speciation analysis revealed that 94-98% of the total arsenate in cells of T. pyriformis could be transformed to monomethylarsonic acid, dimethylarsinic acid and a small proportion of arsenite after 18 h of arsenate exposure, thus indicating the major detoxification pathway by arsenic oxidation/reduction and biomethylation. Finally, comparative proteomic analysis unveiled significant changes in the expression of multiple proteins involved in anti-oxidation, sugar and energy metabolism, proteolysis, and signal transduction. Our results revealed multiple pathways of arsenate detoxification in T. pyriformis, and indicated that protozoa may play important roles in the biogeochemical cycles of arsenic.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 10⁴ cell·ml⁻¹ or more, the effect of shaking was absent. However, with inoculates of 10³ or 10² cell·ml⁻¹, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.